Optical measurements are routinely used to detect and quantify the presence of species in chemical and biological samples. The most common forms of optical measurement are absorption, fluorescence, and luminescence. In absorbance measurements, the degree of attenuation caused by optical absorption is related to the concentration of the species of interest. Fluorescence measurements involve exciting the sample via short-wavelength optical radiation and measuring the optical power of longer-wavelength fluorescence. Luminescence measurements rely on the measurement of light emitted by the sample without the presence of excitation light, most often caused by a chemical reaction.
In modern analytical instruments, small sample volumes are arranged in a multiple-well plate known as a microplate, usually comprised of 96, 384 or 1536 individual wells. A typical instrument optically scans the wells and detects optical power. The optical power is then related to the sample concentration using a known functional relationship. Fluorescence is most commonly used as a sensitive means to detect very low sample concentrations and maintain a wide dynamic range.
Despite the large number of patents pertaining to fluorometric instruments, little attention has been paid to the elimination of stray excitation light, which can cause significant signal degradation. Stray light can arise from a multitude of sources, including lens surfaces, microplate window surfaces, and scattering within the optical system. However, the dominant source of stray light in a microplate fluorometer is usually the liquid meniscus at the sample-air interface. This source of stray light is particularly difficult to reject since the meniscus can vary in position (due to different sample volumes) and curvature (due to different surface properties). Most importantly, a spatially extended beam impinging on the meniscus results in a wide angular bandwidth upon reflection, making it difficult to achieve rejection by simple baffling alone. One patent that has attempted to improve upon the prior art in this regard is listed below.
U.S. Pat. No. 6,316,774, titled “Optical System for a Scanning Fluorometer”, discloses an optical instrument for the measurement of absorbance and fluorescence from microplates. The patent teaches a method of measuring fluorescence from above and below a microplate well using elliptical reflective mirrors that are arranged in a manner in which the collection of excitation beam scatter is reduced. Unfortunately, the invention only succeeds in eliminating the collection of scattered excitation light for a very limited range of sample volumes and meniscus curvatures.
It would be very advantageous to provide an optical system that could broadly be used to reduce interference arising from liquid scattering of excitation light from a liquid meniscus in any type of optical system used for optical analysis of liquids contained in vessels, which advantageously could be integrated into a microplate reader or automated assay instrument in order to provide a compact assembly for sensitive fluorescence measurements that avoids or reduces the interference of scattered light from the liquid meniscus.